GCMS analysis of valproic acid metabolites and the effects of salicylate on valproic acid metabolism

An assay has been developed for the simultaneous quantitative analysis of VPA and twelve of its metabolites namely 2-ene VPA, 3-ene VPA, 4-ene VPA, (E)-2,4-diene VPA, (E,E)-2,3'-diene VPA, 3-OH VPA, 4-OH VPA, 5-OH VPA, 3-keto VPA, 4-keto VPA, 2-propylsuccinic acid and 2-propylglutaric acid. The assay is accomplished in a single run using the Hewlett-Packard 5987A GC-MS. VPA and its metabolites were measured using selected ion monitoring of the characteristic (M-57)⁺ ions of the tertbutyldimethylsilyl (tBDMS) derivatives. The 4-OH VPA was measured as the underivatized ϒ-lactone. [²H₆]-VPA, [²H₃]-2-ene VPA and 3-octanone served as internal standards. In a typical assay 1 mL of urine or serum is adjusted to pH 13 to hydrolyze the conjugates, then acidified to pH 2 and extracted with ethyl acetate. The solvent is then dried, concentrated to 200 μL and derivatized with tBDMS reagent. The chromatographic run time with a 25 m x 0.32 mm I.D. bonded phase OV 1701 capillary column was 22 minutes. All metabolites were well resolved and extracts of control serum or urine showed no interferences. The tBDMS and TMS derivatives of VPA metabolites were compared. Mixed tBDMS-TMS derivatives were also investigated. This assay has been applied to the analysis of urine and serum samples from pediatric patients on VPA therapy. The effects of acetyl salicylic acid (ASA) on the metabolism of VPA were investigated. Urine samples of six pediatric patients under valproic acid therapy and a normal adult subject taking valproic acid were obtained before and after administration of ASA. The analyses showed that the excretion of conjugated and unchanged VPA was enhanced in all subjects following administration of ASA. The 3-keto VPA, the second major urinary metabolite after VPA-glucuronide, was markedly reduced. The average excretion of 2-ene VPA and 3-OH VPA was also lowered. These results suggest that the β-oxidation pathway which includes 2-ene VPA, 3-OH VPA and 3-keto VPA was inhibited by salicylate at some stage prior to the formation of 2-ene VPA. Since serum samples of the normal subject were available the kinetics of VPA metabolites before and after administration of ASA were determined. It was found that the formation clearance, the fraction metabolized and the elimination clearance of the β-oxidation metabolites were decreased following ASA administration. These results confirmed the urinary data obtained with the seven subjects. Salicylate was found to inhibit the β-oxidation of VPA. Other metabolic pathways of VPA were not significantly affected by ASA administration. The mechanism by which salicylate affects the β-oxidation of valproic acid is discussed.Pharmaceutical Sciences, Faculty ofGraduat